Multiple scattering X-ray absorption studies of Zn²⁺ binding sites in bacterial photosynthetic reaction centers

¹ Dipartimento di Fisica, Universita' di Bologna
² Dipartimento di Biologia, Universita' di Bologna
³ Istituto per i Processi Chimico-Fisici, CNR, Bari
⁴ Dipartimento di Chimica, Universita' di Bari
⁵ Dipartimento di Biochimica, Universita' di Bologna

^* To whom correspondence should be addressed. E-mail: ventur{at}alma.unibo.it.

Submitted on August 3, 2004
Revised on September 8, 2004
Accepted on 15 December 2004

	Abstract

Binding of transition metal ions to the reaction center (RC) protein of the photosynthetic bacterium Rhodobacter sphaeroides has been previously shown to slow light-induced electron and proton transfer to the secondary quinone acceptor molecule, Q_B. On the basis of X-ray diffraction (XRD) at 2.5 Å resolution a site, formed by AspH124, HisH126 and HisH128, has been identified at the protein surface which binds Cd²⁺ or Zn²⁺. Using Zn K-edge X-ray absorption fine structure (XAFS) spectroscopy we report here on the local structure of Zn²⁺ ions bound to purified RC complexes embedded into polyvinyl alcohol films. XAFS data were analysed by combining ab-initio simulations and multiparameter fitting; structural contributions up to the fourth coordination shell and multiple scattering paths (involving three atoms) have been included. Results for complexes characterized by a Zn to RC stoichiometry close to 1 indicate that Zn²⁺ binds two O and two N atoms in the first coordination shell. Higher shell contributions are consistent with a binding cluster formed by two His, one Asp residue and a water molecule. Analysis of complexes characterized by approximately 2 Zn ions per RC reveals a second structurally distinct binding site, involving one O and three N atoms, not belonging to a His residue. The local structure obtained for the higher affinity site nicely fits the coordination geometry proposed on the basis of XRD data, but detects a significant contraction of the first shell. Two possible locations of the second new binding site at the cytoplasmic surface of the RC are proposed.

Key Words: Rhodobacter sphaeroides, X-ray absorption spectroscopy, Zn binding sites, local structure, multiple scattering, reaction center

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